Cationic lipids for use in lipid nanoparticles

ABSTRACT

Compounds are provided having the following structure:or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein a, b, c, d, G1, G2, L1, L2, R1a, R1b, R2a, R2b, R3a, R3b, R4a, R4b, R5, R6, R7, R8 and X are as defined herein. Use of the compounds as a component of lipid nanoparticle formulations for delivery of a therapeutic agent, nanoparticles comprising the compounds and methods for their use and preparation are also provided.

BACKGROUND Technical Field

The present disclosure generally relates to novel cationic lipids thatcan be used in combination with other lipid components, such as neutrallipids, cholesterol and polymer conjugated lipids, to form lipidnanoparticles to facilitate the intracellular delivery of therapeuticagents, such as nucleic acids (e.g., oligonucleotides, messenger RNA),both in vitro and in vivo.

Description of the Related Art

There are many challenges associated with the delivery of nucleic acidsto affect a desired response in a biological system. Nucleic acid basedtherapeutics have enormous potential but there remains a need for moreeffective delivery of nucleic acids to appropriate sites within a cellor organism in order to realize this potential. Therapeutic nucleicacids include, e.g., messenger RNA (mRNA), antisense oligonucleotides,ribozymes, DNAzymes, plasmids, immune stimulating nucleic acids,antagomir, antimir, mimic, supermir, and aptamers. Some nucleic acids,such as mRNA or plasmids, can be used to effect expression of specificcellular products as would be useful in the treatment of, for example,diseases related to a deficiency of a protein or enzyme. The therapeuticapplications of translatable nucleotide delivery are extremely broad asconstructs can be synthesized to produce any chosen protein sequence,whether or not indigenous to the system. The expression products of thenucleic acid can augment existing levels of protein, replace missing ornon-functional versions of a protein, or introduce new protein andassociated functionality in a cell or organism.

Some nucleic acids, such as miRNA inhibitors, can be used to effectexpression of specific cellular products that are regulated by miRNA aswould be useful in the treatment of, for example, diseases related todeficiency of protein or enzyme. The therapeutic applications of miRNAinhibition are extremely broad as constructs can be synthesized toinhibit one or more miRNA that would in turn regulate the expression ofmRNA products. The inhibition of endogenous miRNA can augment itsdownstream target endogenous protein expression and restore properfunction in a cell or organism as a means to treat disease associated toa specific miRNA or a group of miRNA.

Other nucleic acids can down-regulate intracellular levels of specificmRNA and, as a result, down-regulate the synthesis of the correspondingproteins through processes such as RNA interference (RNAi) orcomplementary binding of antisense RNA. The therapeutic applications ofantisense oligonucleotide and RNAi are also extremely broad, sinceoligonucleotide constructs can be synthesized with any nucleotidesequence directed against a target mRNA. Targets may include mRNAs fromnormal cells, mRNAs associated with disease-states, such as cancer, andmRNAs of infectious agents, such as viruses. To date, antisenseoligonucleotide constructs have shown the ability to specificallydown-regulate target proteins through degradation of the cognate mRNA inboth in vitro and in vivo models. In addition, antisense oligonucleotideconstructs are currently being evaluated in clinical studies.

However, two problems currently face the use of oligonucleotides intherapeutic contexts. First, free RNAs are susceptible to nucleasedigestion in plasma. Second, free RNAs have limited ability to gainaccess to the intracellular compartment where the relevant translationmachinery resides. Lipid nanoparticles formed from cationic lipids withother lipid components, such as neutral lipids, cholesterol, PEG,PEGylated lipids, and oligonucleotides have been used to blockdegradation of the RNAs in plasma and facilitate the cellular uptake ofthe oligonucleotides.

There remains a need for improved cationic lipids and lipidnanoparticles for the delivery of oligonucleotides. Preferably, theselipid nanoparticles would provide optimal drug:lipid ratios, protect thenucleic acid from degradation and clearance in serum, be suitable forsystemic or local delivery, and provide intracellular delivery of thenucleic acid. In addition, these lipid-nucleic acid particles should bewell-tolerated and provide an adequate therapeutic index, such thatpatient treatment at an effective dose of the nucleic acid is notassociated with unacceptable toxicity and/or risk to the patient. Thepresent disclosure provides these and related advantages.

BRIEF SUMMARY

In brief, the present disclosure provides lipid compounds, includingstereoisomers, pharmaceutically acceptable salts or tautomers thereof,which can be used alone or in combination with other lipid componentssuch as neutral lipids, charged lipids, steroids (including for example,all sterols) and/or their analogs, and/or polymer conjugated lipids toform lipid nanoparticles for the delivery of therapeutic agents. In someinstances, the lipid nanoparticles are used to deliver nucleic acidssuch as antisense and/or messenger RNA. Methods for use of such lipidnanoparticles for treatment or prevention (e.g., vaccination) of variousdiseases or conditions, such as those caused by infectious entitiesand/or insufficiency of a protein, are also provided.

In one embodiment, compounds having the following structure (I) areprovided:

or a pharmaceutically acceptable salt, tautomer, or stereoisomerthereof, wherein a, b, c, d, G¹, G², L¹, L², R^(1a), R^(1b), R^(2a),R^(2b), R^(3a), R^(3b), R^(4a), R^(4b), R⁵, R⁶, R⁷, R⁸ and X are asdefined herein.

Lipid nanoparticles (LNPs) comprising one or more of the compounds ofstructure (I) and a therapeutic agent, and pharmaceutical compositionscomprising the same, are also provided. In some embodiments, thenanoparticles further comprise one or more components selected fromneutral lipids, charged lipids, steroids, and polymer conjugated lipids.Such LNPs are useful for delivery of the therapeutic agent, for examplefor treatment of a disease or vaccination against a viral pathogen.

These and other aspects of the disclosure will be apparent uponreference to the following detailed description.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments of thedisclosure. However, one skilled in the art will understand that thedisclosure may be practiced without these details.

The present disclosure is based, in part, upon the discovery of novelcationic (amino) lipids that provide advantages when used in lipidnanoparticles for the in vivo delivery of an active or therapeutic agentsuch as a nucleic acid into a cell of a mammal. In particular,embodiments of the present disclosure provide nucleic acid-lipidnanoparticle compositions comprising one or more of the novel cationiclipids described herein that provide increased activity of the nucleicacid and improved tolerability of the compositions in vivo, resulting ina significant increase in the therapeutic index as compared to nucleicacid-lipid nanoparticle compositions previously described. In otherembodiments, the disclosed lipids, and lipid nanoparticles comprisingthe same, have increased safety and/or tolerability when used fordelivery of active agents, such as nucleic acids.

In particular embodiments, the present disclosure provides novelcationic lipids that enable the formulation of improved compositions forthe in vitro and in vivo delivery of mRNA and/or other oligonucleotides.In some embodiments, these improved lipid nanoparticle compositions areuseful for expression of protein encoded by mRNA. In other embodiments,these improved lipid nanoparticles compositions are useful forupregulation of endogenous protein expression by delivering miRNAinhibitors targeting one specific miRNA or a group of miRNA regulatingone target mRNA or several mRNA. In other embodiments, these improvedlipid nanoparticle compositions are useful for down-regulating (e.g.,silencing) the protein levels and/or mRNA levels of target genes. Insome other embodiments, the lipid nanoparticles are also useful fordelivery of mRNA and plasmids for expression of transgenes. In yet otherembodiments, the lipid nanoparticle compositions are useful for inducinga pharmacological effect resulting from expression of a protein, e.g.,increased production of red blood cells through the delivery of asuitable erythropoietin mRNA, or protection against infection throughdelivery of mRNA encoding for a suitable antigen or antibody.

The lipid nanoparticles and compositions of embodiments of the presentdisclosure may be used for a variety of purposes, including the deliveryof encapsulated or associated (e.g., complexed) therapeutic agents suchas nucleic acids to cells, both in vitro and in vivo. Accordingly,embodiments of the present disclosure provide methods of treating orpreventing diseases or disorders in a subject in need thereof bycontacting the subject with a lipid nanoparticle that encapsulates or isassociated with a suitable therapeutic agent, wherein the lipidnanoparticle comprises one or more of the novel cationic lipidsdescribed herein.

As described herein, embodiments of the lipid nanoparticles of thepresent disclosure are particularly useful for the delivery of nucleicacids, including, e.g., mRNA, antisense oligonucleotide, plasmid DNA,microRNA (miRNA), miRNA inhibitors (antagomirs/antimirs),messenger-RNA-interfering complementary RNA (micRNA), DNA, multivalentRNA, dicer substrate RNA, complementary DNA (cDNA), etc. Therefore, thelipid nanoparticles and compositions of certain embodiments of thepresent disclosure may be used to induce expression of a desired proteinboth in vitro and in vivo by contacting cells with a lipid nanoparticlecomprising one or more novel cationic lipids described herein, whereinthe lipid nanoparticle encapsulates or is associated with a nucleic acidthat is expressed to produce the desired protein (e.g., a messenger RNAor plasmid encoding the desired protein) or inhibit processes thatterminate expression of mRNA (e.g., miRNA inhibitors). In certainembodiments, the protein expressed by the nucleic acid is an antigen,and the LNPs thus induce an immune response (e.g., vaccination).Alternatively, the lipid nanoparticles and compositions of embodimentsof the present disclosure may be used to decrease the expression oftarget genes and proteins both in vitro and in vivo by contacting cellswith a lipid nanoparticle comprising one or more novel cationic lipidsdescribed herein, wherein the lipid nanoparticle encapsulates or isassociated with a nucleic acid that reduces target gene expression(e.g., an antisense oligonucleotide or small interfering RNA (siRNA)).The lipid nanoparticles and compositions of embodiments of the presentdisclosure may also be used for co-delivery of different nucleic acids(e.g., mRNA and plasmid DNA) separately or in combination, such as maybe useful to provide an effect requiring co-localization of differentnucleic acids (e.g., mRNA encoding for a suitable gene modifying enzymeand DNA segment(s) for incorporation into the host genome).

Nucleic acids for use with embodiments of this disclosure may beprepared according to any available technique. For mRNA, the primarymethodology of preparation is, but not limited to, enzymatic synthesis(also termed in vitro transcription) which currently represents the mostefficient method to produce long sequence-specific mRNA. In vitrotranscription describes a process of template-directed synthesis of RNAmolecules from an engineered DNA template comprised of an upstreambacteriophage promoter sequence (e.g., including but not limited to thatfrom the T7, T3 and SP6 coliphage) linked to a downstream sequenceencoding the gene of interest. Template DNA can be prepared for in vitrotranscription from a number of sources with appropriate techniques whichare well known in the art including, but not limited to, plasmid DNA andpolymerase chain reaction amplification (see Linpinsel, J. L and Conn,G. L., General protocols for preparation of plasmid DNA template andBowman, J. C., Azizi, B., Lenz, T. K., Ray, P., and Williams, L. D. inRNA in vitro transcription and RNA purification by denaturing PAGE inRecombinant and in vitro RNA syntheses Methods v. 941 Conn G. L. (ed),New York, N.Y. Humana Press, 2012).

Transcription of the RNA occurs in vitro using the linearized DNAtemplate in the presence of the corresponding RNA polymerase andadenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates(rNTPs) under conditions that support polymerase activity whileminimizing potential degradation of the resultant mRNA transcripts. Invitro transcription can be performed using a variety of commerciallyavailable kits including, but not limited to RiboMax Large Scale RNAProduction System (Promega), MegaScript Transcription kits (LifeTechnologies), as well as with commercially available reagents includingRNA polymerases and rNTPs. The methodology for in vitro transcription ofmRNA is well known in the art. (see, e.g., Losick, R., 1972, In vitrotranscription, Ann Rev Biochem v. 41 409-46; Kamakaka, R. T. and Kraus,W. L. 2001. In Vitro Transcription. Current Protocols in Cell Biology.2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B., (2010) Synthesis ofRNA by In Vitro Transcription in RNA in Methods in Molecular Biology v.703 (Neilson, H. Ed), New York, N.Y. Humana Press, 2010; Brunelle, J. L.and Green, R., 2013, Chapter Five—In vitro transcription from plasmid orPCR-amplified DNA, Methods in Enzymology v. 530, 101-114; all of whichare incorporated herein by reference).

The desired in vitro transcribed mRNA is then purified from theundesired components of the transcription or associated reactions(including unincorporated rNTPs, protein enzyme, salts, short RNAoligos, etc.). Techniques for the isolation of the mRNA transcripts arewell known in the art. Well known procedures include phenol/chloroformextraction or precipitation with either alcohol (ethanol, isopropanol)in the presence of monovalent cations or lithium chloride. Additional,non-limiting examples of purification procedures which can be usedinclude size exclusion chromatography (Lukaysky, P. J. and Puglisi, J.D., 2004, Large-scale preparation and purification ofpolyacrylamide-free RNA oligonucleotides, RNA v. 10, 889-893),silica-based affinity chromatography and polyacrylamide gelelectrophoresis (Bowman, J. C., Azizi, B., Lenz, T. K., Ray, P., andWilliams, L. D. in RNA in vitro transcription and RNA purification bydenaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941Conn G. L. (ed), New York, N.Y. Humana Press, 2012). Purification can beperformed using a variety of commercially available kits including, butnot limited to SV Total Isolation System (Promega) and In VitroTranscription Cleanup and Concentration Kit (Norgen Biotek).

Furthermore, while reverse transcription can yield large quantities ofmRNA, the products can contain a number of aberrant RNA impuritiesassociated with undesired polymerase activity which may need to beremoved from the full-length mRNA preparation. These include short RNAsthat result from abortive transcription initiation as well asdouble-stranded RNA (dsRNA) generated by RNA-dependent RNA polymeraseactivity, RNA-primed transcription from RNA templates andself-complementary 3′ extension. It has been demonstrated that thesecontaminants with dsRNA structures can lead to undesiredimmunostimulatory activity through interaction with various innateimmune sensors in eukaryotic cells that function to recognize specificnucleic acid structures and induce potent immune responses. This inturn, can dramatically reduce mRNA translation since protein synthesisis reduced during the innate cellular immune response. Therefore,additional techniques to remove these dsRNA contaminants have beendeveloped and are known in the art including but not limited toscaleable HPLC purification (see, e.g., Kariko, K., Muramatsu, H.,Ludwig, J. And Weissman, D., 2011, Generating the optimal mRNA fortherapy: HPLC purification eliminates immune activation and improvestranslation of nucleoside-modified, protein-encoding mRNA, Nucl AcidRes, v. 39 e142; Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K.,HPLC Purification of in vitro transcribed long RNA in SyntheticMessenger RNA and Cell Metabolism Modulation in Methods in MolecularBiology v. 969 (Rabinovich, P. H. Ed), 2013). HPLC purified mRNA hasbeen reported to be translated at much greater levels, particularly inprimary cells and in vivo.

A significant variety of modifications have been described in the artwhich are used to alter specific properties of in vitro transcribedmRNA, and improve its utility. These include, but are not limited tomodifications to the 5′ and 3′ termini of the mRNA. Endogenouseukaryotic mRNA typically contain a cap structure on the 5′-end of amature molecule which plays an important role in mediating binding ofthe mRNA Cap Binding Protein (CBP), which is in turn responsible forenhancing mRNA stability in the cell and efficiency of mRNA translation.Therefore, highest levels of protein expression are achieved with cappedmRNA transcripts. The 5′-cap contains a 5′-5′-triphosphate linkagebetween the 5′-most nucleotide and guanine nucleotide. The conjugatedguanine nucleotide is methylated at the N7 position. Additionalmodifications include methylation of the ultimate and penultimate most5′-nucleotides on the 2′-hydroxyl group.

Multiple distinct cap structures can be used to generate the 5′-cap ofin vitro transcribed synthetic mRNA. 5′-capping of synthetic mRNA can beperformed co-transcriptionally with chemical cap analogs (i.e., cappingduring in vitro transcription). For example, the Anti-Reverse Cap Analog(ARCA) cap contains a 5′-5′-triphosphate guanine-guanine linkage whereone guanine contains an N7 methyl group as well as a 3′-O-methyl group.However, up to 20% of transcripts remain uncapped during thisco-transcriptional process and the synthetic cap analog is not identicalto the 5′-cap structure of an authentic cellular mRNA, potentiallyreducing translatability and cellular stability. Alternatively,synthetic mRNA molecules may also be enzymatically cappedpost-transcriptionally. These may generate a more authentic 5′-capstructure that more closely mimics, either structurally or functionally,the endogenous 5′-cap which have enhanced binding of cap bindingproteins, increased half-life and reduced susceptibility to 5′endonucleases and/or reduced 5′ decapping. Numerous synthetic 5′-capanalogs have been developed and are known in the art to enhance mRNAstability and translatability (see, e.g., Grudzien-Nogalska, E.,Kowalska, J., Su, W., Kuhn, A. N., Slepenkov, S. V., Darynkiewicz, E.,Sahin, U., Jemielity, J., and Rhoads, R. E., Synthetic mRNAs withsuperior translation and stability properties in Synthetic Messenger RNAand Cell Metabolism Modulation in Methods in Molecular Biology v. 969(Rabinovich, P. H. Ed), 2013).

On the 3′-terminus, a long chain of adenine nucleotides (poly-A tail) isnormally added to mRNA molecules during RNA processing. Immediatelyafter transcription, the 3′ end of the transcript is cleaved to free a3′ hydroxyl to which poly-A polymerase adds a chain of adeninenucleotides to the RNA in a process called polyadenylation. The poly-Atail has been extensively shown to enhance both translational efficiencyand stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly (A),poly (A) binding protein and the regulation of mRNA stability, TrendsBio Sci v. 14 373-377; Guhaniyogi, J. And Brewer, G., 2001, Regulationof mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M.And Regnier, P., 2002, The poly (A) tail of mRNAs: Bodyguard ineukaryotes, scavenger in bacteria, Cell, v. 111, 611-613).

Poly (A) tailing of in vitro transcribed mRNA can be achieved usingvarious approaches including, but not limited to, cloning of a poly (T)tract into the DNA template or by post-transcriptional addition usingPoly (A) polymerase. The first case allows in vitro transcription ofmRNA with poly (A) tails of defined length, depending on the size of thepoly (T) tract, but requires additional manipulation of the template.The latter case involves the enzymatic addition of a poly (A) tail to invitro transcribed mRNA using poly (A) polymerase which catalyzes theincorporation of adenine residues onto the 3′termini of RNA, requiringno additional manipulation of the DNA template, but results in mRNA withpoly(A) tails of heterogeneous length. 5′-capping and 3′-poly (A)tailing can be performed using a variety of commercially available kitsincluding, but not limited to Poly (A) Polymerase Tailing kit(EpiCenter), mMESSAGE mMACHINE T7 Ultra kit and Poly (A) Tailing kit(Life Technologies) as well as with commercially available reagents,various ARCA caps, Poly (A) polymerase, etc.

In addition to 5′ cap and 3′ poly adenylation, other modifications ofthe in vitro transcripts have been reported to provide benefits asrelated to efficiency of translation and stability. It is well known inthe art that pathogenic DNA and RNA can be recognized by a variety ofsensors within eukaryotes and trigger potent innate immune responses.The ability to discriminate between pathogenic and self DNA and RNA hasbeen shown to be based, at least in part, on structure and nucleosidemodifications since most nucleic acids from natural sources containmodified nucleosides. In contrast, in vitro synthesized RNA lacks thesemodifications, thus rendering it immunostimulatory which in turn caninhibit effective mRNA translation as outlined above. The introductionof modified nucleosides into in vitro transcribed mRNA can be used toprevent recognition and activation of RNA sensors, thus mitigating thisundesired immunostimulatory activity and enhancing translation capacity(see, e.g., Kariko, K. And Weissman, D. 2007, Naturally occurringnucleoside modifications suppress the immunostimulatory activity of RNA:implication for therapeutic RNA development, Curr Opin Drug DiscovDevel, v. 10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko,K., In vitro transcription of long RNA containing modified nucleosidesin Synthetic Messenger RNA and Cell Metabolism Modulation in Methods inMolecular Biology v. 969 (Rabinovich, P. H. Ed), 2013; Kariko, K.,Muramatsu, H., Welsh, F. A., Ludwig, J., Kato, H., Akira, S., Weissman,D., 2008, Incorporation of Pseudouridine Into mRNA Yields SuperiorNonimmunogenic Vector With Increased Translational Capacity andBiological Stability, Mol Ther v. 16, 1833-1840). The modifiednucleosides and nucleotides used in the synthesis of modified RNAs canbe prepared monitored and utilized using general methods and proceduresknown in the art. A large variety of nucleoside modifications areavailable that may be incorporated alone or in combination with othermodified nucleosides to some extent into the in vitro transcribed mRNA(see, e.g., US 2012/0251618). In vitro synthesis of nucleoside-modifiedmRNA has been reported to have reduced ability to activate immunesensors with a concomitant enhanced translational capacity.

Other components of mRNA which can be modified to provide benefit interms of translatability and stability include the 5′ and 3′untranslated regions (UTR). Optimization of the UTRs (favorable 5′ and3′ UTRs can be obtained from cellular or viral RNAs), either both orindependently, have been shown to increase mRNA stability andtranslational efficiency of in vitro transcribed mRNA (see, e.g., Pardi,N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription oflong RNA containing modified nucleosides in Synthetic Messenger RNA andCell Metabolism Modulation in Methods in Molecular Biology v. 969(Rabinovich, P. H. Ed), 2013).

In addition to mRNA, other nucleic acid payloads may be used for thisdisclosure. For oligonucleotides, methods of preparation include but arenot limited to chemical synthesis and enzymatic, chemical cleavage of alonger precursor, in vitro transcription as described above, etc.Methods of synthesizing DNA and RNA nucleotides are widely used and wellknown in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotidesynthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.:IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis:methods and applications, Methods in Molecular Biology, v. 288 (Clifton,N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporatedherein by reference).

For plasmid DNA, preparation for use with embodiments of this disclosurecommonly utilizes, but is not limited to, expansion and isolation of theplasmid DNA in vitro in a liquid culture of bacteria containing theplasmid of interest. The presence of a gene in the plasmid of interestthat encodes resistance to a particular antibiotic (penicillin,kanamycin, etc.) allows those bacteria containing the plasmid ofinterest to selectively grow in antibiotic-containing cultures. Methodsof isolating plasmid DNA are widely used and well known in the art (see,e.g., Heilig, J., Elbing, K. L. and Brent, R., (2001), Large-ScalePreparation of Plasmid DNA, Current Protocols in Molecular Biology,41:11:1.7:1.7.1-1.7.16; Rozkov, A., Larsson, B., Gillström, S.,Björnestedt, R. and Schmidt, S. R., (2008), Large-scale production ofendotoxin-free plasmids for transient expression in mammalian cellculture, Biotechnol. Bioeng., 99: 557-566; and U.S. Pat. No. 6,197,553B1). Plasmid isolation can be performed using a variety of commerciallyavailable kits including, but not limited to Plasmid Plus (Qiagen),GenJET plasmid MaxiPrep (Thermo), and PureYield MaxiPrep (Promega) kitsas well as with commercially available reagents.

Various exemplary embodiments of the cationic lipids of the presentdisclosure, lipid nanoparticles and compositions comprising the same,and their use to deliver active (e.g., therapeutic agents), such asnucleic acids, to modulate gene and protein expression, are described infurther detail below.

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open andinclusive sense, that is, as “including, but not limited to.”

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present disclosure. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this disclosure belongs. As used in the specification andclaims, the singular form “a,” “an” and “the” include plural referencesunless the context clearly dictates otherwise.

The phrase “induce expression of a desired protein” refers to theability of a nucleic acid to increase expression of the desired protein.To examine the extent of protein expression, a test sample (e.g., asample of cells in culture expressing the desired protein) or a testmammal (e.g., a mammal such as a human or an animal) model such as arodent (e.g., mouse) or a non-human primate (e.g., monkey) model iscontacted with a nucleic acid (e.g., nucleic acid in combination with alipid of the present disclosure). Expression of the desired protein inthe test sample or test animal is compared to expression of the desiredprotein in a control sample (e.g., a sample of cells in cultureexpressing the desired protein) or a control mammal (e.g., a mammal suchas a human or an animal) model such as a rodent (e.g., mouse) ornon-human primate (e.g., monkey) model that is not contacted with oradministered the nucleic acid. When the desired protein is present in acontrol sample or a control mammal, the expression of a desired proteinin a control sample or a control mammal may be assigned a value of 1.0.In particular embodiments, inducing expression of a desired protein isachieved when the ratio of desired protein expression in the test sampleor the test mammal to the level of desired protein expression in thecontrol sample or the control mammal is greater than 1, for example,about 1.1, 1.5, 2.0. 5.0 or 10.0. When a desired protein is not presentin a control sample or a control mammal, inducing expression of adesired protein is achieved when any measurable level of the desiredprotein in the test sample or the test mammal is detected. One ofordinary skill in the art will understand appropriate assays todetermine the level of protein expression in a sample, for example dotblots, northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, and phenotypic assays, or assaysbased on reporter proteins that can produce fluorescence or luminescenceunder appropriate conditions.

The phrase “inhibiting expression of a target gene” refers to theability of a nucleic acid to silence, reduce, or inhibit the expressionof a target gene. To examine the extent of gene silencing, a test sample(e.g., a sample of cells in culture expressing the target gene) or atest mammal (e.g., a mammal such as a human or an animal) model such asa rodent (e.g., mouse) or a non-human primate (e.g., monkey) model iscontacted with a nucleic acid that silences, reduces, or inhibitsexpression of the target gene. Expression of the target gene in the testsample or test animal is compared to expression of the target gene in acontrol sample (e.g., a sample of cells in culture expressing the targetgene) or a control mammal (e.g., a mammal such as a human or an animal)model such as a rodent (e.g., mouse) or non-human primate (e.g., monkey)model that is not contacted with or administered the nucleic acid. Theexpression of the target gene in a control sample or a control mammalmay be assigned a value of 100%. In particular embodiments, silencing,inhibition, or reduction of expression of a target gene is achieved whenthe level of target gene expression in the test sample or the testmammal relative to the level of target gene expression in the controlsample or the control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%,60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Inother words, the nucleic acids are capable of silencing, reducing, orinhibiting the expression of a target gene by at least about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% in a test sample or a test mammal relative to thelevel of target gene expression in a control sample or a control mammalnot contacted with or administered the nucleic acid. Suitable assays fordetermining the level of target gene expression include, withoutlimitation, examination of protein or mRNA levels using techniques knownto those of skill in the art, such as, e.g., dot blots, northern blots,in situ hybridization, ELISA, immunoprecipitation, enzyme function, aswell as phenotypic assays known to those of skill in the art.

An “effective amount” or “therapeutically effective amount” of an activeagent or therapeutic agent such as a therapeutic nucleic acid is anamount sufficient to produce the desired effect, e.g., an increase orinhibition of expression of a target sequence in comparison to thenormal expression level detected in the absence of the nucleic acid. Anincrease in expression of a target sequence is achieved when anymeasurable level is detected in the case of an expression product thatis not present in the absence of the nucleic acid. In the case where theexpression product is present at some level prior to contact with thenucleic acid, an in increase in expression is achieved when the foldincrease in value obtained with a nucleic acid such as mRNA relative tocontrol is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000,5000, 10000, or greater. Inhibition of expression of a target gene ortarget sequence is achieved when the value obtained with a nucleic acidsuch as antisense oligonucleotide relative to the control is about 95%,90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expression of atarget gene or target sequence include, e.g., examination of protein orRNA levels using techniques known to those of skill in the art such asdot blots, northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, fluorescence, or luminescence ofsuitable reporter proteins, as well as phenotypic assays known to thoseof skill in the art.

The term “nucleic acid” as used herein refers to a polymer containing atleast two deoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form and includes DNA, RNA, and hybrids thereof. DNA maybe in the form of antisense molecules, plasmid DNA, cDNA, PCR products,or vectors. RNA may be in the form of small hairpin RNA (shRNA),messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA,dicer substrate RNA or viral RNA (vRNA), and combinations thereof.Nucleic acids include nucleic acids containing known nucleotide analogsor modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, and which have similarbinding properties as the reference nucleic acid. Examples of suchanalogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2′-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions), alleles, orthologs, single nucleotide polymorphisms, andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka etal., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell.Probes, 8:91-98 (1994)). “Nucleotides” contain a sugar deoxyribose (DNA)or ribose (RNA), a base, and a phosphate group. Nucleotides are linkedtogether through the phosphate groups. “Bases” include purines andpyrimidines, which further include natural compounds adenine, thymine,guanine, cytosine, uracil, inosine, and natural analogs, and syntheticderivatives of purines and pyrimidines, which include, but are notlimited to, modifications which place new reactive groups such as, butnot limited to, amines, alcohols, thiols, carboxylates, andalkylhalides.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises partial length or entire length coding sequencesnecessary for the production of a polypeptide or precursor polypeptide.

“Gene product,” as used herein, refers to a product of a gene such as anRNA transcript or a polypeptide.

The term “lipid” refers to a group of organic compounds that include,but are not limited to, esters of fatty acids and are generallycharacterized by being poorly soluble in water, but soluble in manyorganic solvents. They are usually divided into at least three classes:(1) “simple lipids,” which include fats and oils as well as waxes; (2)“compound lipids,” which include phospholipids and glycolipids; and (3)“derived lipids” such as steroids.

A “steroid” is a compound comprising the following carbon skeleton:

Non-limiting examples of steroids include cholesterol, and the like.

A “cationic lipid” refers to a lipid capable of being positivelycharged. Exemplary cationic lipids include one or more amine group(s)which bear the positive charge.

Preferred cationic lipids are ionizable such that they can exist in apositively charged or neutral form depending on pH. The ionization ofthe cationic lipid affects the surface charge of the lipid nanoparticleunder different pH conditions. This charge state can influence plasmaprotein absorption, blood clearance, and tissue distribution (Semple, S.C., et al., Adv. Drug Deliv Rev 32:3-17 (1998)) as well as the abilityto form endosomolytic non-bilayer structures (Hafez, I. M., et al., GeneTher 8:1188-1196 (2001)) critical to the intracellular delivery ofnucleic acids.

The term “polymer conjugated lipid” refers to a molecule comprising botha lipid portion and a polymer portion. An example of a polymerconjugated lipid is a pegylated lipid. The term “pegylated lipid” refersto a molecule comprising both a lipid portion and a polyethylene glycolportion. Pegylated lipids are known in the art and include1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) andthe like.

The term “neutral lipid” refers to any of a number of lipid species thatexist either in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids include, but are not limited to,phosphotidylcholines such as 1,2-Distearoyl-sn-glycero-3-phosphocholine(DSPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),phophatidylethanolamines such as1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelins(SM), ceramides, steroids such as sterols and their derivatives. Neutrallipids may be synthetic or naturally derived.

The term “charged lipid” refers to any of a number of lipid species thatexist in either a positively charged or negatively charged formindependent of the pH within a useful physiological range, e.g., pH ˜3to pH ˜9. Charged lipids may be synthetic or naturally derived. Examplesof charged lipids include phosphatidylserines, phosphatidic acids,phosphatidylglycerols, phosphatidylinositols, sterol hemi succinates,dialkyl trimethylammonium-propanes, (e.g., DOTAP, DOTMA), dialkyldimethylaminopropanes, ethyl phosphocholines, dimethylaminoethanecarbamoyl sterols (e.g., DC-Chol).

The term “lipid nanoparticle” refers to particles having at least onedimension on the order of nanometers (e.g., 1-1,000 nm) which includeone or more of the compounds of structure (I) or other specifiedcationic lipids. In some embodiments, lipid nanoparticles comprising thedisclosed cationic lipids (e.g., compounds of structure (I)) areincluded in a formulation that can be used to deliver an active agent ortherapeutic agent, such as a nucleic acid (e.g., mRNA) to a target siteof interest (e.g., cell, tissue, organ, tumor, and the like). In someembodiments, the lipid nanoparticles comprise a compound of structure(I) and a nucleic acid. Such lipid nanoparticles typically comprise acompound of structure (I) and one or more excipient selected fromneutral lipids, charged lipids, steroids, and polymer conjugated lipids.In some embodiments, the active agent or therapeutic agent, such as anucleic acid, may be encapsulated in the lipid portion of the lipidnanoparticle or an aqueous space enveloped by some or all of the lipidportion of the lipid nanoparticle, thereby protecting it from enzymaticdegradation or other undesirable effects induced by the mechanisms ofthe host organism or cells, e.g., an adverse immune response.

In various embodiments, the lipid nanoparticles have a mean diameter offrom about 30 nm to about 150 nm, from about 40 nm to about 150 nm, fromabout 50 nm to about 150 nm, from about 60 nm to about 130 nm, fromabout 70 nm to about 110 nm, from about 70 nm to about 100 nm, fromabout 80 nm to about 100 nm, from about 90 nm to about 100 nm, fromabout 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm.In some embodiments, the lipid nanoparticles are substantiallynon-toxic. In certain embodiments, nucleic acids, when present in thelipid nanoparticles, are resistant in aqueous solution to degradationwith a nuclease. Lipid nanoparticles comprising nucleic acids and theirmethod of preparation are disclosed in, e.g., U.S. Patent Pub. Nos.2004/0142025, 2007/0042031 and PCT Pub. Nos. WO 2013/016058 and WO2013/086373, the full disclosures of which are herein incorporated byreference in their entirety for all purposes.

As used herein, “lipid encapsulated” refers to a lipid nanoparticle thatprovides an active agent or therapeutic agent, such as a nucleic acid(e.g., mRNA), with full encapsulation, partial encapsulation, or both.In an embodiment, the nucleic acid (e.g., mRNA) is fully encapsulated inthe lipid nanoparticle.

As used herein, the term “aqueous solution” refers to a compositioncomprising water.

“Serum-stable” in relation to nucleic acid-lipid nanoparticles meansthat the nucleotide is not significantly degraded after exposure to aserum or nuclease assay that would significantly degrade free DNA orRNA. Suitable assays include, for example, a standard serum assay, aDNAse assay, or an RNAse assay.

“Systemic delivery,” as used herein, refers to delivery of a therapeuticproduct that can result in a broad exposure of an active agent within anorganism. Some techniques of administration can lead to the systemicdelivery of certain agents, but not others. Systemic delivery means thata useful, preferably therapeutic, amount of an agent is exposed to mostparts of the body. Systemic delivery of lipid nanoparticles can be byany means known in the art including, for example, intravenous,intraarterial, subcutaneous, and intraperitoneal delivery. In someembodiments, systemic delivery of lipid nanoparticles is by intravenousdelivery.

“Local delivery,” as used herein, refers to delivery of an active agentdirectly to a target site within an organism. For example, an agent canbe locally delivered by direct injection into a disease site such as atumor, other target site such as a site of inflammation, or a targetorgan such as the liver, heart, pancreas, kidney, and the like. Localdelivery can also include topical applications or localized injectiontechniques such as intramuscular, subcutaneous, or intradermalinjection. Local delivery does not preclude a systemic pharmacologicaleffect.

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, which is saturated, andhaving, for example, from one to twenty-four carbon atoms (C₁-C₂₄alkyl), four to twenty carbon atoms (C₄-C₂₀ alkyl), six to sixteencarbon atoms (C₆-C₁₆ alkyl), six to nine carbon atoms (C₆-C₉ alkyl), oneto fifteen carbon atoms (C₁-C₁₅ alkyl), one to twelve carbon atoms(C₁-C₁₂ alkyl), one to eight carbon atoms (C₁-C₈ alkyl) or one to sixcarbon atoms (C₁-C₆ alkyl) and which is attached to the rest of themolecule by a single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl(iso propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl),3-methylhexyl, 2-methylhexyl, and the like. Unless stated otherwisespecifically in the specification, an alkyl group is substituted orunsubstituted.

“Alkylene” refers to a straight or branched divalent hydrocarbon chainlinking the rest of the molecule to a radical group, consisting solelyof carbon and hydrogen, which is saturated, and having, for example,from one to twenty-four carbon atoms (C₁-C₂₄ alkylene), one to fifteencarbon atoms (C₁-C₁₅ alkylene), one to twelve carbon atoms (C₁-C₁₂alkylene), one to eight carbon atoms (C₁-C₈ alkylene), one to six carbonatoms (C₁-C₆ alkylene), two to four carbon atoms (C₂-C₄ alkylene), oneto two carbon atoms (C₁-C₂ alkylene), e.g., methylene, ethylene,propylene, n-butylene, and the like. The alkylene chain is attached tothe rest of the molecule through a single bond and to the radical groupthrough a single bond. The points of attachment of the alkylene chain tothe rest of the molecule and to the radical group can be through onecarbon or any two carbons within the chain. Unless stated otherwisespecifically in the specification, an alkylene chain is substituted orunsubstituted.

“Alkene” and “alkenylene” refer to an alkyl and alkylene, respectively,comprising at least one carbon-carbon double bond. Alkenes andalkenylenes include the same number of carbon atoms as alkyl andalkylene as defined above, except that alkenes and alkenylenes mustinclude at least two carbons. Unless stated otherwise specifically inthe specification, alkenes and alkenylenes are substituted orunsubstituted.

The term “substituted” used herein means any of the above groups (e.g.,alkyl or alkylene) wherein at least one hydrogen atom is replaced by abond to a non-hydrogen atom such as, but not limited to: a halogen atomsuch as F, Cl, Br, or I; oxo groups (═O); hydroxyl groups (—OH);carboxyl groups —(CO₂H); C₁-C₁₂ alkyl groups; —(C═O)OR′; —O(C═O)R′;—C(═O)R′; —OR′; —S(O)_(x)R′; —S—SR′; —C(═O)SR′; —SC(═O)R′; —NR′R′;—NRC(═O)R′; —C(═O)NR′R′; —NRC(═O)NR′R′; —OC(═O)NR′R′; —NR′C(═O)OR′;—NR′S(O)_(x)NR′R′; —NRS(O)_(x)R′; and —S(O)_(x)NR′R, wherein: R′ is, ateach occurrence, independently H or C₁-C₁₅ alkyl and x is 0, 1 or 2. Insome embodiments the substituent is a C₁-C₁₂ alkyl group. In otherembodiments, the substituent is a halo group, such as fluoro. In otherembodiments, the substituent is an oxo group. In other embodiments, thesubstituent is a hydroxyl group. In other embodiments, the substituentis an alkoxy group (—OR). In other embodiments, the substituent is acarboxyl group. In other embodiments, the substituent is an amine group(—NR′R′).

“Optional” or “optionally” (e.g., optionally substituted) means that thesubsequently described event of circumstances may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances in which it does not. For example, “optionallysubstituted alkyl” means that the alkyl radical may or may not besubstituted and that the description includes both substituted alkylradicals and alkyl radicals having no substitution.

The disclosure disclosed herein is also meant to encompass allpharmaceutically acceptable compounds of the compound of structure (I)being isotopically-labelled by having one or more atoms replaced by anatom having a different atomic mass or mass number. Examples of isotopesthat can be incorporated into the disclosed compounds include isotopesof hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine,and iodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³¹P,³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I, and ¹²⁵I, respectively. These radiolabeledcompounds could be useful to help determine or measure the effectivenessof the compounds, by characterizing, for example, the site or mode ofaction, or binding affinity to pharmacologically important site ofaction. Certain isotopically-labelled compounds of structure (I), (IA)or (IB), for example, those incorporating a radioactive isotope, areuseful in drug and/or substrate tissue distribution studies. Theradioactive isotopes tritium, i.e., ³H, and carbon-14, i.e., ¹⁴C, areparticularly useful for this purpose in view of their ease ofincorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e., ²H, mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining substrate receptor occupancy. Isotopically-labeled compoundsof structure (I) can generally be prepared by conventional techniquesknown to those skilled in the art or by processes analogous to thosedescribed in the Preparations and Examples as set out below using anappropriate isotopically-labeled reagent in place of the non-labeledreagent previously employed.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

“Mammal” includes humans and both domestic animals such as laboratoryanimals and household pets (e.g., cats, dogs, swine, cattle, sheep,goats, horses, rabbits), and non-domestic animals such as wildlife andthe like.

“Pharmaceutically acceptable carrier, diluent or excipient” includeswithout limitation any adjuvant, carrier, excipient, glidant, sweeteningagent, diluent, preservative, dye/colorant, flavor enhancer, surfactant,wetting agent, dispersing agent, suspending agent, stabilizer, isotonicagent, solvent, or emulsifier which has been approved by the UnitedStates Food and Drug Administration as being acceptable for use inhumans or domestic animals.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but are not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecyl sulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroaceticacid, undecylenic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as ammonia,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

Often crystallizations produce a solvate of a compound of the disclosure(i.e., a compound of structure (I)). As used herein, the term “solvate”refers to an aggregate that comprises one or more molecules of acompound of the disclosure with one or more molecules of solvent. Thesolvent may be water, in which case the solvate may be a hydrate.Alternatively, the solvent may be an organic solvent. Thus, thecompounds of the present disclosure may exist as a hydrate, including amonohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate,tetrahydrate and the like, as well as the corresponding solvated forms.Solvates of compound of the disclosure may be true solvates, while inother cases the compound of the disclosure may merely retainadventitious water or be a mixture of water plus some adventitioussolvent.

A “pharmaceutical composition” refers to a formulation of a compound ofthe disclosure and a medium generally accepted in the art for thedelivery of the biologically active compound to mammals, e.g., humans.Such a medium includes all pharmaceutically acceptable carriers,diluents or excipients therefor.

“Effective amount” or “therapeutically effective amount” refers to thatamount of a compound of the disclosure which, when administered to amammal, preferably a human, is sufficient to effect treatment in themammal, preferably a human. The amount of a lipid nanoparticle of thedisclosure which constitutes a “therapeutically effective amount” willvary depending on the compound, the condition and its severity, themanner of administration, and the age of the mammal to be treated, butcan be determined routinely by one of ordinary skill in the art havingregard to his own knowledge and to this disclosure.

“Treating” or “treatment” as used herein covers the treatment of thedisease or condition of interest in a mammal, preferably a human, havingthe disease or condition of interest, and includes:

-   -   (i) preventing the disease or condition from occurring in a        mammal, in particular, when such mammal is predisposed to the        condition but has not yet been diagnosed as having it;    -   (ii) inhibiting the disease or condition, i.e., arresting its        development;    -   (iii) relieving the disease or condition, i.e., causing        regression of the disease or condition; or    -   (iv) relieving the symptoms resulting from the disease or        condition, i.e., relieving pain without addressing the        underlying disease or condition. As used herein, the terms        “disease” and “condition” may be used interchangeably or may be        different in that the particular malady or condition may not        have a known causative agent (so that etiology has not yet been        worked out) and it is therefore not yet recognized as a disease        but only as an undesirable condition or syndrome, wherein a more        or less specific set of symptoms have been identified by        clinicians.

The compounds of the disclosure, or their pharmaceutically acceptablesalts may contain one or more stereocenters and may thus give rise toenantiomers, diastereomers, and other stereoisomeric forms that may bedefined, in terms of absolute stereochemistry, as (R)- or (S)- or, as(D)- or (L)- for amino acids. The present disclosure is meant to includeall such possible isomers, as well as their racemic and optically pureforms. Optically active (+) and (−), (R)- and (S)-, or (D)- and(L)-isomers may be prepared using chiral synthons or chiral reagents, orresolved using conventional techniques, for example, chromatography andfractional crystallization. Conventional techniques for thepreparation/isolation of individual enantiomers include chiral synthesisfrom a suitable optically pure precursor or resolution of the racemate(or the racemate of a salt or derivative) using, for example, chiralhigh pressure liquid chromatography (HPLC). When the compounds describedherein contain olefinic double bonds or other centers of geometricasymmetry, and unless specified otherwise, it is intended that thecompounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present disclosure contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers,”which refers to two stereoisomers whose molecules are non-superimposablemirror images of one another.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. The present disclosure includestautomers of any said compounds.

Compounds

In an aspect, the disclosure provides novel lipid compounds which arecapable of combining with other lipid components such as neutral lipids,charged lipids, steroids and/or polymer conjugated-lipids to form lipidnanoparticles with therapeutic agents, such as oligonucleotides. Withoutwishing to be bound by theory, it is thought that these lipidnanoparticles shield the therapeutic agent from degradation in the serumand provide for effective delivery of the therapeutic agent to cells invitro and in vivo.

In one embodiment, the compounds have the following structure (I):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein:

-   -   G¹ and G² are each independently C₁-C₆ alkylene;    -   L¹ and L² are each independently —O(C═O)— or —(C═O)O—;    -   R^(1a) and R^(1b) are, at each occurrence, independently        either: (a) H or C₁-C₁₂ alkyl; or (b) R^(1a) is H or C₁-C₁₂        alkyl, and R^(1b) together with the carbon atom to which it is        bound is taken together with an adjacent R^(1b) and the carbon        atom to which it is bound to form a carbon-carbon double bond;    -   R^(2a) and R^(2b) are, at each occurrence, independently        either: (a) H or C₁-C₁₂ alkyl; or (b) R^(2a) is H or C₁-C₁₂        alkyl, and R^(2b) together with the carbon atom to which it is        bound is taken together with an adjacent R^(2b) and the carbon        atom to which it is bound to form a carbon-carbon double bond;

R^(1a) and R^(ab) are, at each occurrence, independently either (a): Hor C₁-C₁₂ alkyl; or (b) R^(3a) is H or C₁-C₁₂ alkyl, and R^(ab) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(3b) and the carbon atom to which it is bound to form acarbon-carbon double bond;

R^(4a) and R^(4b) are, at each occurrence, independently either: (a) Hor C₁-C₁₂ alkyl; or (b) R^(4a) is H or C₁-C₁₂ alkyl, and R^(4b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(4b) and the carbon atom to which it is bound to form acarbon-carbon double bond;

R⁵ and R⁶ are each independently H or methyl;

-   -   R⁷ is —O(C═O)R¹⁰, —(C═O)OR¹⁰, —NR⁹(C═O)R¹⁰ or —(C═O)NR⁹R¹⁰;    -   R⁸ is OH, —N(R¹¹)(C═O)R¹², —(C═O)NR¹¹R¹², —NR¹¹R¹², —(C═O)OR¹²        or —O(C═O)R¹²;    -   R⁹ is H or C₁-C₁₅ alkyl;    -   R¹⁰ is C₁-C₁₅ alkyl;    -   R¹¹ is H or C₁-C₆ alkyl;    -   R¹² is C₁-C₆ alkyl;    -   X is —(C═O)— or a direct bond; and    -   a, b, c and d are each independently an integer from 1 to 24;

wherein each alkyl and alkylene and is independently optionallysubstituted.

In other embodiments, the compound has one of the following structures(IA) or (IB):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

In certain embodiments, G¹ is C₂-C₃ alkylene. In different embodiments,G¹ is C₄-C₆ alkylene. For example, in various embodiments, G¹ is C₂, C₃,C₄, C₅ or C₆ alkylene.

In other embodiments, G² is C₂-C₄ alkylene, for example C₂-C₃ alkyleneor C₃-C₄ alkylene. In some embodiments, G² is C₂, C₃ or C₄ alkylene.

In various different embodiments, X is —(C═O)—, while in differentembodiments X is a direct bond.

In any of the foregoing embodiments, R⁷ is —O(C═O)R¹⁰ or —(C═O)OR¹⁰. Incertain of these embodiments, R¹⁰ is linear C₁-C₁₅ alkyl, such as linearC₆-C₁₀ alkyl. In other such embodiments, R¹⁰ is methyl or R¹⁰ isbranched C₂-C₁₅ alkyl, such as branched C₁₀-C₁₅ alkyl.

In still other of the foregoing embodiments, R⁷ is —NR⁹(C═O) or—(C═O)NR⁹R¹⁰. In some of these embodiments, R⁹ is H. In other of theseembodiments, R⁹ and R¹⁰ are each independently C₆-C₁₀ alkyl.

In other embodiments, at least one occurrence of R^(1a) and R^(1b),R^(1a) is H or C₁-C₁₂ alkyl, and R^(1b) together with the carbon atom towhich it is bound is taken together with an adjacent R^(1b) and thecarbon atom to which it is bound to form a carbon-carbon double bond.

In more embodiments, for at least one occurrence of R^(4a) and R^(4b),R^(4a) is H or C₁-C₁₂ alkyl, and R^(4b) together with the carbon atom towhich it is bound is taken together with an adjacent R^(4b) and thecarbon atom to which it is bound to form a carbon-carbon double bond.

In still other embodiments, for at least one occurrence of R^(2a) andR^(2b), R^(2a) is H or C₁-C₁₂ alkyl, and R^(2b) together with the carbonatom to which it is bound is taken together with an adjacent R^(2b) andthe carbon atom to which it is bound to form a carbon-carbon doublebond.

In other embodiments, for at least one occurrence of R^(1a) and R^(3b),R^(3a) is H or C₁-C₁₂ alkyl, and R^(3b) together with the carbon atom towhich it is bound is taken together with an adjacent R^(3b) and thecarbon atom to which it is bound to form a carbon-carbon double bond.

In various of the foregoing embodiments, R^(1a), R^(1b), R^(2a), R^(2b),R^(3a), R^(3b), R^(4a) and R^(4b) are, at each occurrence, independentlyH or C₁-C₁₂ alkyl. In other embodiments, R^(2a), R^(2b), R^(3a) andR^(3b) are, at each occurrence, H. For example, in certain embodimentR^(1a) and R^(4a) are, at each occurrence, H. In different embodiments,at least one of R^(1b) and R^(4b) is C₁-C₈ alkyl. For example, in someembodiments C₁-C₈ alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl,iso-butyl, tert-butyl, n-hexyl or n-octyl.

In other more specific embodiments,

or both, independently has one of the following structures:

In more embodiments, a, b, c and d are each independently an integerfrom 2 to 12. In more embodiments, a, b, c and d are each independentlyan integer from 4 to 10, 5 to 10, 6 to 10, 4 to 9, 5 to 9 or 6 to 9. Instill different embodiments, b and c are independently 5, 6, 7, 8, 9 or10.

In some embodiments, one of R⁵ or R⁶ is methyl. In other embodiments,each of R⁵ and R⁶ is methyl.

In some embodiments, R⁸ is OH.

In other embodiments, R⁸ is —N(R¹¹)(C═O)R¹². In different embodiments,R⁸ is —(C═O)NR¹¹R¹². In more different embodiments, R⁸ is NR¹¹R¹². Insome of these foregoing embodiments, R¹¹ and R¹² are each independentlyH or C₁-C₈ alkyl. In other of these embodiments, R¹¹ and R¹² are eachindependently H or C₁-C₃ alkyl. For example, in some embodiments theC₁-C₈ alkyl or C₁-C₃ alkyl is unsubstituted or substituted withhydroxyl. In other different such embodiments, R¹¹ and R¹² are eachmethyl.

In other embodiments of the compound of structure (I), R⁸ is —(C═O)OR¹²,while in different embodiments R⁸ is —O(C═O)R¹².

In specific embodiments of any of the foregoing compounds, R⁸ has one ofthe following structures:

In various different embodiments, the disclosure provides a compoundhaving one of the structures set forth in Table 1 below, or apharmaceutically acceptable salt or tautomer thereof.

TABLE 1 Representative Compounds No. Structure I-1

I-2

I-3

I-4

I-5

I-6

I-7

I-8

I-9

I-10

I-11

I-12

I-13

I-14

I-15

I-16

I-17

I-18

I-19

I-20

I-21

I-22

I-23

It is understood that any embodiment of the compounds of structure (I),as set forth above, and any specific substituent and/or variable in thecompound of structure (I), as set forth above, may be independentlycombined with other embodiments and/or substituents and/or variables ofcompounds of structure (I) to form embodiments of the disclosures notspecifically set forth above. In addition, in the event that a list ofsubstituents and/or variables is listed for any particular R group, Ggroup, L group or variable a, b, c, d or n, in a particular embodimentand/or claim, it is understood that each individual substituent and/orvariable may be deleted from the particular embodiment and/or claim andthat the remaining list of substituents and/or variables will beconsidered to be within the scope of the disclosure.

It is understood that in the present description, combinations ofsubstituents and/or variables of the depicted formulae are permissibleonly if such contributions result in stable compounds.

In some embodiments, lipid nanoparticles comprising a compound ofstructure (I) are provided. The lipid nanoparticles optionally includeexcipients selected from a neutral lipid, a steroid and a polymerconjugated lipid.

In some embodiments, compositions comprising any one or more of thecompounds of structure (I) and a therapeutic agent are provided. Forexample, in some embodiments, the compositions comprise any of thecompounds of structure (I) and a therapeutic agent and one or moreexcipient selected from neutral lipids, steroids and polymer conjugatedlipids. Other pharmaceutically acceptable excipients and/or carriers arealso included in various embodiments of the compositions.

In some embodiments, the neutral lipid is selected from DSPC, DPPC,DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid isDSPC. In various embodiments, the molar ratio of the compound to theneutral lipid ranges from about 2:1 to about 8:1.

In various embodiments, the compositions further comprise a steroid orsteroid analogue. In certain embodiments, the steroid or steroidanalogue is cholesterol. In some of these embodiments, the molar ratioof the compound to cholesterol ranges from about 5:1 to 1:1.

In various embodiments, the polymer conjugated lipid is a pegylatedlipid. For example, some embodiments include a pegylated diacylglycerol(PEG-DAG) such as1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), apegylated phosphatidylethanoloamine (PEG-PE), a PEG succinatediacylglycerol (PEG-S-DAG) such as4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate(PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEGdialkoxypropylcarbamate such asω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. Invarious embodiments, the molar ratio of the compound to the pegylatedlipid ranges from about 100:1 to about 20:1.

In some embodiments, the composition comprises a pegylated lipid havingthe following structure (II):

-   -   or a pharmaceutically acceptable salt, tautomer or stereoisomer        thereof, wherein:

R⁸ and R⁹ are each independently a straight or branched, saturated orunsaturated alkyl chain containing from 10 to 30 carbon atoms, whereinthe alkyl chain is optionally interrupted by one or more ester bonds;and

-   -   w has a mean value ranging from 30 to 60.

In some embodiments, R⁸ and R⁹ are each independently straight,saturated alkyl chains containing from 12 to 16 carbon atoms. In otherembodiments, the average w ranges from about 42 to 55, for example about49.

In some embodiments of the foregoing composition, the therapeutic agentcomprises a nucleic acid. For example, in some embodiments, the nucleicacid is selected from antisense and messenger RNA. In some of theforegoing embodiments, the composition comprises a lipid nanoparticle.

Some related embodiments provide a lipid nanoparticle comprising thecompound of any one of the foregoing embodiments (e.g., a compound ofstructure (I)). In certain embodiments, the lipid nanoparticle furthercomprises a therapeutic agent (e.g., a nucleic acid such as antisenseand messenger RNA).

In some embodiments, the lipid nanoparticle further comprises one ormore excipient selected from neutral lipids, steroids and polymerconjugated lipids. In some embodiments, the neutral lipids are selectedfrom DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In more specificembodiments, the neutral lipid is DSPC.

In some more specific embodiments, the molar ratio of the compound tothe neutral lipid ranges from about 2:1 to about 8:1. In someembodiments, the steroid is cholesterol. In some embodiments, the molarratio of the compound to cholesterol ranges from 5:1 to 1:1.

In certain embodiments, the polymer conjugated lipid is pegylated lipid.In certain more specific embodiments, the molar ratio of the compound topegylated lipid ranges from about 100:1 to about 20:1.

In some embodiments, the pegylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG,PEG-cer or a PEG dialkyoxypropylcarbamate. In other embodiments, thepegylated lipid has the following structure (II):

-   -   or a pharmaceutically acceptable salt, tautomer or stereoisomer        thereof, wherein:

R⁸ and R⁹ are each independently a straight or branched, saturated orunsaturated alkyl chain containing from 10 to 30 carbon atoms, whereinthe alkyl chain is optionally interrupted by one or more ester bonds;and

-   -   w has a mean value ranging from 30 to 60.

In some more specific embodiments of structure (II), R⁸ and R⁹ are eachindependently straight, saturated alkyl chains containing from 12 to 16carbon atoms. In more specific embodiments, the average w is about 49.

In other different embodiments, the disclosure is directed to a methodfor administering a therapeutic agent to a patient in need thereof, themethod comprising preparing or providing any of the foregoingcompositions and administering the composition to the patient

For the purposes of administration, embodiments of the compounds of thepresent disclosure (typically in the form of lipid nanoparticles incombination with a therapeutic agent) may be administered as a rawchemical or may be formulated as pharmaceutical compositions.Pharmaceutical compositions of embodiments of the present disclosurecomprise a compound of structure (I) and one or more pharmaceuticallyacceptable carrier, diluent or excipient. In some embodiments, thecompound of structure (I) is present in the composition in an amountwhich is effective to form a lipid nanoparticle and deliver thetherapeutic agent, e.g., for treating a particular disease or conditionof interest. Appropriate concentrations and dosages can be readilydetermined by one skilled in the art.

Administration of the compositions of embodiments of the disclosure canbe carried out via any of the accepted modes of administration of agentsfor serving similar utilities. The pharmaceutical compositions ofembodiments of the disclosure may be formulated into preparations insolid, semi-solid, liquid or gaseous forms, such as tablets, capsules,powders, granules, ointments, solutions, suspensions, suppositories,injections, inhalants, gels, microspheres, and aerosols. Typical routesof administering such pharmaceutical compositions include, withoutlimitation, oral, topical, transdermal, inhalation, parenteral,sublingual, buccal, rectal, vaginal, and intranasal. The term parenteralas used herein includes subcutaneous injections, intravenous,intramuscular, intradermal, intrasternal injection or infusiontechniques. Pharmaceutical compositions of embodiments of the disclosureare formulated so as to allow the active ingredients contained thereinto be bioavailable upon administration of the composition to a patient.Compositions that will be administered to a subject or patient in someembodiments take the form of one or more dosage units, where forexample, a tablet may be a single dosage unit, and a container of acompound of an embodiments of the disclosure in aerosol form may hold aplurality of dosage units. Actual methods of preparing such dosage formsare known, or will be apparent, to those skilled in this art; forexample, see Remington: The Science and Practice of Pharmacy, 20thEdition (Philadelphia College of Pharmacy and Science, 2000). In someembodiments, the composition to be administered will, in any event,contain a therapeutically effective amount of a compound of thedisclosure, or a pharmaceutically acceptable salt thereof, for treatmentof a disease or condition of interest in accordance with the teachingsof this disclosure.

A pharmaceutical composition of embodiments of the disclosure may be inthe form of a solid or liquid. In one aspect, the carrier(s) areparticulate, so that the compositions are, for example, in tablet orpowder form. The carrier(s) may be liquid, with the compositions being,for example, oral syrup, injectable liquid or an aerosol, which isuseful in, for example, inhalatory administration.

When intended for oral administration, the pharmaceutical composition ofcertain embodiments is preferably in either solid or liquid form, wheresemi-solid, semi-liquid, suspension and gel forms are included withinthe forms considered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceuticalcomposition of some embodiments may be formulated into a powder,granule, compressed tablet, pill, capsule, chewing gum, wafer or thelike form. Such a solid composition will typically contain one or moreinert diluents or edible carriers. In addition, one or more of thefollowing may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, gum tragacanth or gelatin;excipients such as starch, lactose or dextrins, disintegrating agentssuch as alginic acid, sodium alginate, Primogel, corn starch and thelike; lubricants such as magnesium stearate or Sterotex; glidants suchas colloidal silicon dioxide; sweetening agents such as sucrose orsaccharin; a flavoring agent such as peppermint, methyl salicylate ororange flavoring; and a coloring agent.

When the pharmaceutical composition of some embodiments is in the formof a capsule, for example, a gelatin capsule, it may contain, inaddition to materials of the above type, a liquid carrier such aspolyethylene glycol or oil.

The pharmaceutical composition of some embodiments may be in the form ofa liquid, for example, an elixir, syrup, solution, emulsion orsuspension. The liquid may be for oral administration or for delivery byinjection, as two examples. When intended for oral administration,preferred composition contain, in addition to a compound of structure(I), one or more of a sweetening agent, preservatives, dye/colorant andflavor enhancer. In a composition intended to be administered byinjection, one or more of a surfactant, preservative, wetting agent,dispersing agent, suspending agent, buffer, stabilizer and isotonicagent may be included.

The liquid pharmaceutical compositions of embodiments of the disclosure,whether they be solutions, suspensions or other like form, may includeone or more of the following adjuvants: sterile diluents such as waterfor injection, saline solution, preferably physiological saline,Ringer's solution, isotonic sodium chloride, fixed oils such assynthetic mono or diglycerides which may serve as the solvent orsuspending medium, polyethylene glycols, glycerin, propylene glycol orother solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose; agents to act ascryoprotectants such as sucrose or trehalose. The parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic. Physiological saline is a preferred adjuvant.An injectable pharmaceutical composition is preferably sterile.

The pharmaceutical composition of embodiments of the disclosure may beintended for topical administration, in which case the carrier maysuitably comprise a solution, emulsion, ointment or gel base. The base,for example, may comprise one or more of the following: petrolatum,lanolin, polyethylene glycols, bee wax, mineral oil, diluents such aswater and alcohol, and emulsifiers and stabilizers. Thickening agentsmay be present in a pharmaceutical composition for topicaladministration. If intended for transdermal administration, thecomposition may include a transdermal patch or iontophoresis device.

The pharmaceutical composition of embodiments of the disclosure mayinclude various materials, which modify the physical form of a solid orliquid dosage unit. For example, the composition may include materialsthat form a coating shell around the active ingredients. The materialsthat form the coating shell are typically inert, and may be selectedfrom, for example, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredients may be encased in a gelatincapsule.

The pharmaceutical composition of embodiments of the disclosure in solidor liquid form may include an agent that binds to the compound of thedisclosure and thereby assists in the delivery of the LNP. Suitableagents that may act in this capacity include a monoclonal or polyclonalantibody, or a protein.

The pharmaceutical composition of embodiments of the disclosure mayconsist of dosage units that can be administered as an aerosol. The termaerosol is used to denote a variety of systems ranging from those ofcolloidal nature to systems consisting of pressurized packages. Deliverymay be by a liquefied or compressed gas or by a suitable pump systemthat dispenses the active ingredients. Aerosols of LNPs of embodimentsof the disclosure may be delivered in single phase, bi-phasic, ortri-phasic systems in order to deliver the active ingredient(s).Delivery of the aerosol includes the necessary container, activators,valves, sub-containers, and the like, which together may form a kit. Oneskilled in the art, without undue experimentation, may determinepreferred aerosols.

The pharmaceutical compositions of embodiments of the disclosure may beprepared by methodology well known in the pharmaceutical art. Forexample, a pharmaceutical composition intended to be administered byinjection can be prepared by combining the lipid nanoparticles of thedisclosure with sterile, distilled water or other carrier so as to forma solution. A surfactant may be added to facilitate the formation of ahomogeneous solution or suspension. Surfactants are compounds thatnon-covalently interact with the compound of the disclosure so as tofacilitate dissolution or homogeneous suspension of the compound in theaqueous delivery system.

The compositions of embodiments of the disclosure, or theirpharmaceutically acceptable salts, are administered in a therapeuticallyeffective amount, which will vary depending upon a variety of factorsincluding the activity of the specific therapeutic agent employed; themetabolic stability and length of action of the therapeutic agent; theage, body weight, general health, sex, and diet of the patient; the modeand time of administration; the rate of excretion; the drug combination;the severity of the particular disorder or condition; and the subjectundergoing therapy.

Compositions of embodiments of the disclosure may also be administeredsimultaneously with, prior to, or after administration of one or moreother therapeutic agents. Such combination therapy includesadministration of a single pharmaceutical dosage formulation of acomposition of embodiments of the disclosure and one or more additionalactive agents, as well as administration of the composition ofembodiments of the disclosure and each active agent in its own separatepharmaceutical dosage formulation. For example, a composition ofembodiments of the disclosure and the other active agent can beadministered to the patient together in a single oral dosage compositionsuch as a tablet or capsule, or each agent administered in separate oraldosage formulations. Where separate dosage formulations are used, thecompounds of embodiments of the disclosure and one or more additionalactive agents can be administered at essentially the same time, i.e.,concurrently, or at separately staggered times, i.e., sequentially;combination therapy is understood to include all these regimens.

Preparation methods for the above compounds and compositions aredescribed herein below and/or known in the art.

It will be appreciated by those skilled in the art that in the processdescribed herein the functional groups of intermediate compounds mayneed to be protected by suitable protecting groups. Such functionalgroups include hydroxy, amino, mercapto and carboxylic acid. Suitableprotecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl(for example, t-butyldimethylsilyl, t-butyldiphenylsilyl ortrimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitableprotecting groups for amino, amidino and guanidino includet-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protectinggroups for mercapto include —C(O)—R″ (where R″ is alkyl, aryl orarylalkyl), p-methoxybenzyl, trityl and the like. Suitable protectinggroups for carboxylic acid include alkyl, aryl or arylalkyl esters.Protecting groups may be added or removed in accordance with standardtechniques, which are known to one skilled in the art and as describedherein. The use of protecting groups is described in detail in Green, T.W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999),3^(rd) Ed., Wiley. As one of skill in the art would appreciate, theprotecting group may also be a polymer resin such as a Wang resin, Rinkresin or 2-chlorotrityl-chloride resin.

It will also be appreciated by those skilled in the art, although suchprotected derivatives of compounds of this disclosure may not possesspharmacological activity as such, they may be administered to a mammaland thereafter metabolized in the body to form compounds of thedisclosure which are pharmacologically active. Such derivatives maytherefore be described as “prodrugs.” All prodrugs of compounds of thisdisclosure are included within the scope of the disclosure.

Furthermore, compounds of embodiments of the disclosure which exist infree base or acid form can be converted to their pharmaceuticallyacceptable salts by treatment with the appropriate inorganic or organicbase or acid by methods known to one skilled in the art. Salts ofcompounds of embodiments of the disclosure can be converted to theirfree base or acid form by standard techniques.

The following General Reaction Scheme 1 illustrates exemplary methods tomake compounds of this disclosure, i.e., compounds of structure (I):

or a pharmaceutically acceptable salt, tautomer, or stereoisomerthereof, wherein a, b, c, d, G¹, G², L¹, L², R^(1a), R^(1b), R^(2a),R^(2b), R^(3a), R^(3b), R^(4a), R^(4b), R⁵, R⁶, R⁷, R⁸ and X are asdefined herein. It is understood that one skilled in the art may be ableto make these compounds by similar methods or by combining other methodsknown to one skilled in the art. It is also understood that one skilledin the art would be able to make, in a similar manner as describedbelow, other compounds of structure (I) not specifically illustratedbelow by using the appropriate starting components and modifying theparameters of the synthesis as needed. In general, starting componentsmay be obtained from sources such as Sigma Aldrich, Lancaster Synthesis,Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. orsynthesized according to sources known to those skilled in the art (see,for example, Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, 5th edition (Wiley, December 2000)) or prepared as describedin this disclosure.

General Reaction Scheme I provides an exemplary method for preparationof a compound of structure (I) (i.e., A5) a, b, c, d, G², L¹, L²,R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R^(4a), compound ofstructure (I) (i.e., A5). a, b, c, d, G¹, L¹, L², R^(1a), R^(1b),R^(2a), R^(2b), R^(3a), R^(3b), R^(4a), R^(4b), R⁵, R⁶, R⁸, R⁹ and R¹⁰in General reaction Scheme 1 are as defined herein, and Z represents anactivated analogue of G¹ sufficient for bond formation with the NH groupof A3 (e.g., an alkene or an alkylene terminating in an aldehyde, acidhalide, acrylate, etc.). Intermediates and reagents (e.g., A1 and A2)needed for preparation of the compounds according to General ReactionScheme I can be purchased or prepared according to the examples below ormethods known by one of ordinary skill in the art.

It should be noted that various alternative strategies for preparationof compounds of structure (I) are available to those of ordinary skillin the art. For example, other compounds of structure (I) wherein can beprepared according to analogous methods using the appropriate startingmaterial. The use of protecting groups as needed and other modificationto the above General Reaction Scheme will be readily apparent to one ofordinary skill in the art.

The following examples are provided for purpose of illustration and notlimitation.

Example 1 Luciferase mRNA In Vivo Evaluation Using the LipidNanoparticle Compositions

Lipid nanoparticles were prepared and tested according to the generalprocedures described in PCT Pub. Nos. WO 2015/199952 and WO 2017/004143,the full disclosures of which are incorporated herein by reference.Briefly, cationic lipid, DSPC, cholesterol and PEG-lipid weresolubilized in ethanol at a molar ratio of about 50:10:38.5:1.5 or about47.5:10:40.8:1.7. Lipid nanoparticles (LNP) were prepared at a totallipid to mRNA weight ratio of approximately 10:1 to 30:1. The mRNA isdiluted to 0.2 mg/mL in 10 to 50 mM citrate or acetate buffer, pH 4.Syringe pumps were used to mix the ethanolic lipid solution with themRNA aqueous solution at a ratio of about 1:5 to 1:3 (vol/vol) withtotal flow rates above 15 mL/min. The ethanol was then removed and theexternal buffer replaced with PBS by dialysis. Finally, the lipidnanoparticles were filtered through a 0.2 μm pore sterile filter. Lipidnanoparticle particle size was approximately 55-95 nm diameter, and insome instances approximately 70-90 nm diameter as determined byquasi-elastic light scattering using a Malvern Zetasizer Nano ZS(Malvern, UK).

Studies were performed in 6-8 week old female C₅₇BL/6 mice (CharlesRiver) or 8-10 week old CD-1 (Harlan) mice (Charles River) according toguidelines established by an institutional animal care committee (ACC)and the Canadian Council on Animal Care (CCAC). Varying doses ofmRNA-lipid nanoparticle were systemically administered by tail veininjection and animals euthanized at a specific time point (e.g., 4hours) post-administration. Liver and spleen were collected inpre-weighed tubes, weights determined, immediately snap frozen in liquidnitrogen and stored at −80° C. until processing for analysis.

For liver, approximately 50 mg was dissected for analyses in a 2 mLFastPrep tubes (MP Biomedicals, Solon Ohio). ¼″ ceramic sphere (MPBiomedicals) is added to each tube and 500 μL of Glo Lysis Buffer—GLB(Promega, Madison Wis.) equilibrated to room temperature is added toliver tissue. Liver tissues were homogenized with the FastPrep24instrument (MP Biomedicals) at 2×6.0 m/s for 15 seconds. Homogenate wasincubated at room temperature for 5 minutes prior to a 1:4 dilution inGLB and assessed using SteadyGlo Luciferase assay system (Promega).Specifically, 50 μL of diluted tissue homogenate was reacted with 50 μLof SteadyGlo substrate, shaken for 10 seconds followed by 5 minuteincubation and then quantitated using a CentroXS³ LB 960 luminometer(Berthold Technologies, Germany). The amount of protein assayed wasdetermined by using the BCA protein assay kit (Pierce, Rockford Ill.).Relative luminescence units (RLU) were then normalized to total μgprotein assayed. To convert RLU to ng luciferase a standard curve wasgenerated with QuantiLum Recombinant Luciferase (Promega).

The FLuc mRNA (L-6107 or L-7202) from Trilink Biotechnologies willexpress a luciferase protein, originally isolated from the firefly,Photinus pyralis. FLuc is commonly used in mammalian cell culture tomeasure both gene expression and cell viability. It emitsbioluminescence in the presence of the substrate, luciferin. This cappedand poly-adenylated mRNA was fully substituted with respect to uridineand/or cytidine nucleosides.

Example 2 Determination of pK_(A) of Formulated Lipids

As described elsewhere, the pK_(a) of formulated cationic lipids iscorrelated with the effectiveness of LNPs for delivery of nucleic acids(see Jayaraman et al, Angewandte Chemie, International Edition (2012),51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176(2010)). The preferred range of pK_(a) is ˜5 to ˜7. The pK_(a) of eachcationic lipid was determined in lipid nanoparticles using an assaybased on fluorescence of 2-(p-toluidino)-6-napthalene sulfonic acid(TNS). Lipid nanoparticles comprising cationiclipid/DSPC/cholesterol/PEG-lipid (50/10/38.5/1.5 mol %) in PBS at aconcentration of 0.4 mM total lipid were prepared using the in-lineprocess as described in Example 1. TNS was prepared as a 100 μM stocksolution in distilled water. Vesicles were diluted to 24 μM lipid in 2mL of buffered solutions containing, 10 mM HEPES, 10 mM IVIES, 10 mMammonium acetate, 130 mM NaCl, where the pH ranged from 2.5 to 11. Analiquot of the TNS solution was added to give a final concentration of 1μM and following vortex mixing fluorescence intensity was measured atroom temperature in a SLM Aminco Series 2 Luminescence Spectrophotometerusing excitation and emission wavelengths of 321 nm and 445 nm. Asigmoidal best fit analysis was applied to the fluorescence data and thepK_(a) was measured as the pH giving rise to half-maximal fluorescenceintensity.

Example 3 Determination of Efficacy of Lipid Nanoparticle FormulationsContaining Various Cationic Lipids Using an In Vivo Luciferase mRNAExpression Rodent Model

Representative compounds of the disclosure shown in Table 2 wereformulated using the following molar ratio: 50% cationic lipid/10%distearoylphosphatidylcholine (DSPC)/38.5% Cholesterol/1.5% PEG lipid2-[2-(ω-methoxy(polyethyleneglycol₂₀₀₀)ethoxy]-N,N-ditetradecylacetamide)or 47.5% cationic lipid/10% DSPC/40.7% Cholesterol/1.8% PEG lipid.Relative activity was determined by measuring luciferase expression inthe liver 4 hours following administration via tail vein injection asdescribed in Example 1. The activity was compared at a dose of 1.0 or0.5 mg mRNA/kg and expressed as ng luciferase/g liver measured 4 hoursafter administration, as described in Example 1. Compound numbers inTable 2 refer to the compound numbers of Table 1.

TABLE 2 Novel Cationic Lipids and Associated Activity Liver Luc Cmp. @1.0 mg/kg No. pKa (ng luc/g liver) Structure I-1 6.11 24031 ± 4777

I-2 6.67 122 ± 18* *determined at 0.3 mg/kg 682 ± 147** **determined at1.0 mg/kg

I-4 6.17 46932 ± 17399

I-5 6.08 11720 ± 2439

I-6 6.22 4093 ± 1036* *determined at 0.3 mg/kg 11290 ± 6455****determined at 1.0 mg/kg

I-14 6.80 669 ± 575* *determined at 0.5 mg/kg

I-15 5.96 21357 ± 15325

I-16 6.25 66763 ± 15823

I-17 6.78 26691 ± 13186

I-18 6.02 40650 ± 11479

I-19 5.95 32706 ± 4621

I-20 6.48 4140 ± 1117* *determined at 0.3 mg/kg 17095 ± 8181****determined at 1.0 mg/kg

I-21 6.40 7723 ± 1714* *determined at 0.3 mg/kg 20223 ± 5982****determined at 1.0 mg/kg

I-22 6.28 5291 ± 1348* *determined at 0.3 mg/kg 17654 ± 8167****determined at 1.0 mg/kg

I-23 6.33 553 ± 153* *determined at 0.5 mg/kg

Example 4 Synthesis of bis(2-butyloctyl)7-((3-(dimethylamino)propyl)(3-(dioctylamino)-3-oxopropyl)amino)tridecanedioate(Compound I-7)

Synthesis of Acryloyl Chloride

Acrylic acid (1.20 g, 16.65 mmol) was dissolved in 20 mL anhydrousdichloromethane. Thionyl chloride (1.98 g, 16.65 mmol) was addeddropwise while stirring under N₂, and the reaction mixture was allowedto heat to reflux for 4 hr. After the completion of reaction, the crudeproduct concentrated to yield a pale yellow liquid that used in the nextstep without further purification.

Synthesis of N,N-Dioctylacrylamide (Intermediate A)

Acryloyl chloride (1.12 g, 12.37 mmol) was added to a cooled solution(0° C.) of dioctylamine in dichloromethane with triethylamine (1 equiv)as a base. The reaction mixture was stirred at 0° C. for 1 h andadditional 1 h at room temperature. The reaction mixture was filteredand the solution obtained was washed with hydrochloric acid (1N HCl),then with sat NaHCO₃ solution and brine. The solvent evaporated underreduced pressure to yield crude product (colorless liquid) which wasused in the following step without further purification.

Synthesis of bis(2-butyloctyl) 7-oxotridecanedioate

To a solution of 2-butyloctane-1-ol (3.85 g, 20.66 mmol),7-oxotridecanedioic acid (1.34 g, 5.17 mmol) and 4-dimethylaminopyridine(DMAP) (1.9 g, 15.55 mmol) in anhydrous DCM was added DCC (4.27 g, 20.69mmol). The resulting mixture was allowed to stir overnight at roomtemperature. The solid (DCU) was then filtered and washed with DCM. Thefiltrate was concentrated. The residue (oil/solid) was purified bycolumn chromatography on silica gel (0-5% ethyl acetate in hexane). Thedesired product was obtained as a colorless oil (2.55 g, 42.86 mmol,83%).

Synthesis of bis(2-butyloctyl)7-((3-(dimethylamino)propyl)amino)tridecanedioate

A solution of 3-(dimethylamino)-1-propylamine (0.09 g, 0.88 mmol) andbis(2-butyloctyl) 7-oxotridecanedioate (0.37 g, 0.63 mmol) in DCE wastreated with sodium triacetoxyborohydride (0.20 g, 0.94 mmol) and AcOH(55 μL, 0.98 mmol) overnight. The solution was washed with diluteaqueous sodium hydroxide solution (1 N NaOH). The organic phase waswashed with brine, dried over anhydrous sodium sulfate, filtered and thesolvent removed. The residue was passed down a small pad of silica gel,washed with a mixture of DCM/MeOH/Et₃N (85:15:1). The filtrate wasconcentrated to give the desired product as a slightly yellow oil (240mg, 0.35 mmol, 56%).

Synthesis of I-7

An EtOH (10 mL) solution of bis(2-butyloctyl)7-((3-(dimethylamino)propyl)amino)tridecanedioate (210 mg, 0.30 mmol)and N,N-Dioctylacrylamide (1.5 eq. 136 mg, 0.46 mmol) was stirred atroom temperature overnight. The reaction mixture was heated to refluxfor 7 days. Upon completion of the reaction, the solvent was removed.The residue was dissolved in a mixture of hexanes and EtOAc (19:1) andwashed with saturated sodium bicarbonate solution, and brine. Theextract was dried over sodium sulfate. The dried extract was filteredthrough a pad of silica gel. The pad was washed with a mixture ofhexane/ethyl acetate/triethylamine (80:20:1). The washing wasconcentrated to give the crude desired product.

The crude product was further purified by flash dry columnchromatography on silica gel (MeOH in chloroform, 0 to 5%). This gavethe desired product as a colorless oil (30 mg, 0.03 mmol, 10%). ¹H NMR(400 MHz, CDCl₃) δ: 3.96 (d, 5.8 Hz, 4H), 3.30-3.16 (m, 4H), 2.74 (t,7.2 Hz, 2H), 2.65-2.24 (m, 17H), 1.80-1.44 (m, 12H), 1.43-1.15 (64H),0.93-0.82 (m, 18H).

Example 5 Synthesis of bis(2-hexyldecyl)7-((4-(dihexylamino)-4-oxobutyl)(2-(dimethylamino)ethyl)amino)tridecanedioate(Compound I-19)

Synthesis of N,N-dihexyl-4-oxobutanamide (Intermediate B)

Butyrolactone (2.51 g, 29.15 mmol) and dihexylamine (5.40 g, 29.13 mmol)were heated for 4 days in a pressure flask at 61° C. The reactionmixture was cooled to room temperature. The crude product was purifiedby column chromatography on silica gel (0% to 5% of MeOH in DCM) toyield N,N-dihexyl-4-hydroxybutanamide as a slightly yellow oil (6.30 g,79%).

N,N-dihexyl-4-hydroxybutanamide (3.00 g, 11.05 mmol) was dissolved inDCM and treated with pyridinium chlorochromate (2.38 g, 11.05 mmol) fortwo hours. Diethyl ether was added and the supernatant filtered throughsilica gel bed. The solvent was removed from the filtrate and resultantoil dissolved in hexane. The suspension was filtered through silica gelbed and the solvent removed. The crude product (colorless liquid) wasused in the following step without further purification.

Synthesis of I-19

A solution of N,N-dihexyl-4-oxobutanamide (0.56 g, 1.97 mmol), andbis(2-hexyldecyl) 7-((2-(dimethylamino)ethyl)amino)tridecanedioate (0.44g, 0.56 mmol, prepare according to procedures of Example 4) in1,2-dichloroethane (10 mL) was stirred for 15 min, after which timesodium triacetoxyborohydride (0.41 g, 1.97 mmol) was added in oneportion and stirred at room temperature for additional 16 hours. Themixture was concentrated. The residue was taken up in a mixture ofhexane and ethyl acetate (96:4) and washed with saturated aqueous NaHCO₃solution and brine. The organic layer was separated, dried overanhydrous sodium sulphate, filtered and evaporated under reducedpressure to obtain colorless oil. The crude product was purified byflash column chromatography on silica gel (MeOH in chloroform, 0 to 5%)to yield the desired product as a colorless oil (260 mg, 0.25 mmol,45%). ¹HNMR (400 MHz, CDCl₃) δ: 3.96 (d, 5.8 Hz, 4H), 3.28 (t-like, 7.7Hz, 2H), 3.20 (t-like, 7.7 Hz, 2H), 2.56-2.47 (m, 2H), 2.44 (t, 6.8 Hz,2H), 2.39-2.20 (m, 15H), 1.74-1.45 (m, 12H), 1.42-1.15 (72H), 0.93-0.84(m, 18H).

Example 6 Synthesis of bis(2-butyloctyl)10-((4-(dihexylamino)-4-oxobutyl)(3-(dimethylamino)propyl)amino)nonadecanedioate(Compound I-21)

Compound I-21 was prepared according to the general procedures ofExample 5 to yield 0.05 g of colorless oil, 0.03 mmol, 32%. ¹HNMR (400MHz, CDCl₃) δ: 3.97 (d, 5.8 Hz, 4H), 3.28 (t-like, 7.6 Hz, 2H), 3.20(t-like, 7.6 Hz, 2H), 2.43-2.23 (m, 13H), 2.20 (s, 6H), 1.75-1.45 (m,14H), 1.40-1.12 (m, 68H), 0.93-0.84 (m, 18H).

Example 7 Synthesis of bis(2-butyloctyl)7-((4-(dihexylamino)-4-oxobutyl)(3-(dimethylamino)propyl)amino)tridecanedioate(Compound I-20)

Compound I-20 was prepared according to the general procedures ofExample 5 to yield 0.06 g of colorless oil, 0.06 mmol, 41%. ¹HNMR (400MHz, CDCl₃) δ: 3.96 (d, 5.8 Hz, 4H), 3.27 (t-like, 7.6 Hz, 2H), 3.19(t-like, 7.6 Hz, 2H), 2.62-2.17 (m, 19H), 1.79-1.43 (m, 14H), 1.42-1.10(m, 56H), 0.95-0.81 (m, 18H).

Example 8 Synthesis of bis(2-butyloctyl)10-(N-(3-(dimethylamino)propyl)-6-methoxy-6-oxohexanamido)nonadecanedioate(Compound I-14)

Adipoyl chloride (0.12 g, 0.68 mmol) in anhydrous benzene (5 mL) wasadded via syringe to a solution of bis(2-butyloctyl)10-((3-(dimethylamino)propyl)amino)nonadecanedioate (0.26 g, 0.34 mmol,prepared according to Example 4), triethylamine (0.3 mL, 2.5 mmol) andDMAP (5 mg) in benzene (10 mL) at RT over the period of 5 min. Themixture was allowed to stir for 2 h and then Methanol (0.5 mL) was addedto remove excess acyl chloride. The resulting mixture was stirred foranother hour and then filtered through a pad of silica gel, washed witha mixture of hexane/EtOAc/Et₃N (70:30:1) and concentrated. The residuewas passed down a silica gel column (0-4% MeOH in DCM gradient),yielding compound I-14 as a colorless oil (0.28 g, 0.30 mmol, 89%).¹HNMR (400 MHz, CDCl₃) δ: 4.52-4.29 (br., estimated 0.3H, due to slowisomerization about amide bond), 3.96 (d, 5.8 Hz, 4H), 3.65 (s, 3H),3.59 (quintet-like, 7.0 Hz, 0.7H), 3.14-3.05 (m, 2H), 2.37-2.24 (m,10H), 2.23-2.18 (m, 6H), 1.73-1.54 (m, 12H), 1.48-1.37 (m, 4H),1.34-1.14 (m, 52H), 0.93-0.83 (m, 12H).

Example 9 Synthesis of bis(2-butyloctyl)10-(N-(2-(dimethylamino)ethyl)-6-methoxy-6-oxohexanamido)nonadecanedioate(Compound I-15)

Compound I-15 was prepared according to the general procedures ofExample 8, to yield 0.18 g of colorless oil, 0.20 mmol, 85%. ¹HNMR (400MHz, CDCl₃) δ: 4.53-4.30 (br., 0.3H, due to slow isomerization aboutamide bond), 3.96 (d, 5.8 Hz, 4H), 3.65 (s, 3H), 3.58 (quintet-like, 7Hz, 0.7H), 3.27-3.15 (m, 2H), 2.46-2.22 (m, 16H), 1.75-1.54 (m, 10H),1.50-1.36 (m, 4H), 1.35-1.09 (m, 52H), 0.94-0.82 (m, 12H).

Example 10 Synthesis of bis(2-hexyldecyl)7-(N-(2-(dimethylamino)ethyl)-6-methoxy-6-oxohexanamido)tridecanedioate(Compound I-16)

Compound I-16 was prepared according to the general procedures ofExample 8 to yield 0.27 g of colorless oil, 0.29 mmol, 81%. ¹HNMR (400MHz, CDCl₃) δ: 4.53-4.30 (br., 0.3H, due to slow isomerization aboutamide bond), 3.99-3.92 (m, 4H), 3.66 (s, 3H), 3.59 (quintet-like, 7.0Hz, 0.7H), 3.28-3.14 (m, 2H), 2.46-2.20 (m, 16H), 1.75-1.53 (m, 10H),1.51-1.36 (m, 4H), 1.35-1.09 (m, 56H), 0.94-0.81 (m, 12H).

Example 11 Synthesis of bis(2-hexyldecyl)7-(N-(3-(dimethylamino)propyl)-8-methoxy-8-oxooctanamido)tridecanedioate(Compound I-17)

Compound I-17 was prepared according to the general procedures ofExample 8 to yield 0.08 g of colorless oil, 0.08 mmol, 75%. ¹HNMR (400MHz, CDCl₃) δ: 4.53-4.30 (br., 0.3H, due to slow isomerization aboutamide bond), 3.99-3.91 (m, 4H), 3.66 (s, 3H), 3.61 (quintet-like, 7.0Hz, 0.7H), 3.15-3.06 (m, 2H), 2.34-2.23 (m, 10H), 2.22 (s, 6H),1.75-1.53 (m, 10H), 1.51-1.38 (m, 4H), 1.37-1.15 (m, 62H), 0.93-0.82 (m,12H).

Example 12 Synthesis of bis(2-hexyldecyl)7-(N-(2-(dimethylamino)ethyl)-8-methoxy-8-oxooctanamido)tridecanedioate(Compound I-18)

Compound I-18 was prepared according to the general procedures ofExample 8 to yield 0.15 g of colorless oil, 0.16 mmol, 79%. ¹HNMR (400MHz, CDCl₃) δ: 4.53-4.30 (br., 0.3H, due to slow isomerization aboutamide bond), 3.99-3.92 (m, 4H), 3.66 (s, 3H), 3.61 (quintet-like, 7.0Hz, 0.7H), 3.26-3.14 (m, 2H), 2.47-2.35 (m, 2H), 2.34-2.20 (m, 14H),1.73-1.53 (m, 8H), 1.51-1.39 (m, 4H), 1.38-1.14 (m, 62H), 0.93-0.82 (m,12H).

Example 13 Synthesis of bis(2-butyloctyl)10-(N-(3-(dimethylamino)propyl)-6-methoxy-6-oxohexanamido)nonadecanedioate(Compound I-1)

Synthesis of Intermediate C

To a solution of acrylic acid (1.1 eq, 8.25 mmol, 594 mg), octanol (1eq, 975 mg, 7.5 mmol) and DMAP (0.4 eq, 3 mmol, 366 mg) in DCM (15 mL)was added DCC (1.4 eq, 10.5 mmol, 2.16 g). The resulting mixture wasstirred at RT for 16 h. The reaction mixture was filtered and thefiltrate concentrated. The residue was taken up in hexanes (50 mL) andloaded on a column of silica gel. The column was washed with hexane (40mL). The fractions were combined, re-loaded on the column and elutedwith a mixture of hexane and ethyl acetate (ca 99:1 or 98:2, 200 mL). Acolorless oil was obtained (986 mg, 71%).

Synthesis of Compound I-1

An EtOH (10 mL) solution of bis(2-butyloctyl)10-((4-(dimethylamino)butyl)amino)nonadecanedioate (1 eq., 220 mg, 0.28mmol, prepared according to general procedures above), and intermediateC (2.75 eq. 0.77 mmol, 140 mg) in a sealed pressure flask was stirred atRT for 4 days under Ar. The reaction mixture was concentrated. Theresidue was purified twice by flash dry column chromatography on silicagel (hexane-EtOAc-Et₃N, 95:5:0 to 80:20:1 and MeOH in chloroform, 0 to5%). The desired product was obtained as colorless oil (68 mg, 0.07mmol, 25%)¹HNMR (400 MHz, CDCl₃) δ: 4.03 (t, 6.9 Hz, 2H), 3.97 (d, 5.8Hz, 4H), 2.69 (t, 7.2 Hz, 2H), 2.38-2.33 (m, 4H), 2.33-2.26 (m, 1H),2.29 (t, 7.5 Hz, 4H), 2.26-2.22 (m, 2H), 2.21 (s, 6H), 1.61(quintet-like, 7.0 Hz, 8H), 1.48-1.08 (70H), 0.92-0.86 (m, 15H).

Example 14 Synthesis of bis(2-butyloctyl)10-((5-(dimethylamino)pentyl)(3-(octyloxy)-3-oxopropyl)amino)nonadecanedioate(Compound I-2)

Compound I-2 was prepared according to the general procedures of Example13 to yield 0.05 g of colorless oil, 0.05 mmol, 10%. ¹HNMR (400 MHz,CDCl₃) δ: 4.03 (t, 6.8 Hz, 2H), 3.96 (d, 5.8 Hz, 4H), 2.69 (t, 7.2 Hz,2H), 2.38-2.21 (m, 17H), 1.61 (quintet-like, 7.0 Hz, 8H), 1.51-1.10 (m,72H), 0.93-0.84 (m, 15H).

Example 15 Synthesis of bis(2-butyloctyl)7-((4-(dimethylamino)butyl)(3-(octyloxy)-3-oxopropyl)amino)tridecanedioate(Compound I-3)

Compound I-3 was prepared according to the general procedures of Example13 to yield 0.01 g of colorless oil, 0.01 mmol, 11%. ¹HNMR (400 MHz,CDCl₃) δ: 4.03 (t, 6.9 Hz, 2H), 3.96 (d, 5.6 Hz, 4H), 2.69 (t, 7.1 Hz,2H), 2.38-2.20 (m, 17H), 1.69-1.56 (m, 10H), 1.48-1.09 (m, 56H),0.92-0.84 (m, 15H).

Example 16 Synthesis of bis(2-hexyldecyl)7-((4-(dimethylamino)butyl)(3-(octyloxy)-3-oxopropyl)amino)tridecanedioate(Compound I-4)

Compound I-4 was prepared according to the general procedures of Example13 to yield 0.05 g of colorless oil, 0.05 mmol, 13%. ¹HNMR (400 MHz,CDCl₃) δ: 4.03 (t, 6.8 Hz, 2H), 3.96 (d, 5.8 Hz, 4H), 2.69 (t, 7.1 Hz,2H), 2.39-2.21 (m, 17H), 1.66-1.09 (m, 82H), 0.88 (t, 7.0 Hz, 15H).

Example 17 Synthesis of bis(2-butyloctyl)10-((4-(dimethylamino)butyl)(octyl)amino)nonadecanedioate (CompoundI-23)

Synthesis of Compound I-23

A solution of octanal (3.5 eq, 0.90 mmol, 115 mg, 0.141 mL) andbis(2-butyloctyl) 10-((4-(dimethylamino)butyl)amino)nonadecanedioate(200 mg, 0.26 mmol, prepared according to general procedures above) in1,2-dichloroethane (5 mL) was stirred for 15 min, after which timesodium triacetoxyborohydride (3.5 eq, 0.9 mmol, 190 mg) was added in oneportion. Stirring was continued at RT for 16 hours. The reaction mixturewas concentrated. The residue was purified twice by flash dry columnchromatography on silica gel (hexane-EtOAc-Et₃N, 95:5:0 to 80:20:1 andMeOH in chloroform, 0 to 5%). The desired product was obtained ascolorless oil (203 mg, 0.23 mmol, 88%)). ¹HNMR (400 MHz, CDCl3) δ: 3.97(d, 5.8 Hz, 4H), 2.40-2.18 (m, 17H), 1.69-1.56 (m, 6H), 1.52-1.10 (m,72H), 0.92-0.86 (m, 15H).

Example 18 Synthesis of bis(2-ethylhexyl)10-((4-(dimethylamino)butyl)(6-((2-hexyldecanoyl)oxy)hexyl)amino)nonadecanedioate(Compound I-22)

Compound I-22 was prepared according to the general procedures ofExample 17 to yield 0.19 g of colorless oil, 0.19 mmol, 80%. ¹HNMR (400MHz, CDCl₃) δ: 4.06 (t, 6.7 Hz, 2H), 3.97 (d, 5.6 Hz, 4H), 2.39-2.26 (m,11H), 2.23 (s, 6H), 1.68-1.10 (m, 83H), 0.94-0.82 (m, 18H).

Example 19 Synthesis of bis(2-butyloctyl)10-((4-(dimethylamino)butyl)(6-((2-hexyldecanoyl)oxy)hexyl)amino)nonadecanedioate(Compound I-5)

Compound I-5 was prepared according to the general procedures of Example17 to yield 0.04 g of colorless oil, 0.04 mmol, 73%. ¹HNMR (400 MHz,CDCl₃) δ: 4.05 (t, 7.0 Hz, 2H), 3.96 (d, 5.8 Hz, 4H), 2.38-2.26 (m,11H), 2.23 (s, 6H), 1.71-1.09 (m, 99H), 0.95-0.82 (m, 18H).

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification are incorporated herein by reference, in their entirety.Aspects of the embodiments can be modified, if necessary to employconcepts of the various patents, applications and publications toprovide yet further embodiments. These and other changes can be made tothe embodiments in light of the above-detailed description. In general,in the following claims, the terms used should not be construed to limitthe claims to the specific embodiments disclosed in the specificationand the claims, but should be construed to include all possibleembodiments along with the full scope of equivalents to which suchclaims are entitled. Accordingly, the claims are not limited by thedisclosure.

1. A compound having a structure of Formula (I):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein: G¹ and G² are each independently C₁-C₆ alkylene; L¹ and L² areeach independently —O(C═O)— or —(C═O)O—; R^(1a) and R^(1b) are, at eachoccurrence, independently either: (a) H or C₁-C₁₂ alkyl; or (b) R^(1a)is H or C₁-C₁₂ alkyl, and R^(1b) together with the carbon atom to whichit is bound is taken together with an adjacent R^(1b) and the carbonatom to which it is bound to form a carbon-carbon double bond; R^(2a)and R^(2b) are, at each occurrence, independently either: (a) H orC₁-C₁₂ alkyl; or (b) R^(2a) is H or C₁-C₁₂ alkyl, and R^(2b) togetherwith the carbon atom to which it is bound is taken together with anadjacent R^(2b) and the carbon atom to which it is bound to form acarbon-carbon double bond; R^(3a) and R^(3b) are, at each occurrence,independently either (a): H or C₁-C₁₂ alkyl; or (b) R^(3a) is H orC₁-C₁₂ alkyl, and R^(3b) together with the carbon atom to which it isbound is taken together with an adjacent R^(3b) and the carbon atom towhich it is bound to form a carbon-carbon double bond; R^(4a) and R^(4b)are, at each occurrence, independently either: (a) H or C₁-C₁₂ alkyl; or(b) R^(4a) is H or C₁-C₁₂ alkyl, and R^(4b) together with the carbonatom to which it is bound is taken together with an adjacent R^(4b) andthe carbon atom to which it is bound to form a carbon-carbon doublebond; R⁵ and R⁶ are each independently H or methyl; R⁷ is —O(C═O)R¹⁰,—(C═O)OR¹⁰, —NR⁹(C═O)R¹⁰ or —(C═O)NR⁹R¹⁰; R⁸ is OH, —N(R¹¹)(C═O)R¹²,—(C═O)NR¹¹R¹², —NR¹¹R¹², —(C═O)OR′² or —O(C═O)R′²; R⁹ is H or C₁-C₁₅alkyl; R¹⁰ is C₁-C₁₅ alkyl; R¹¹ is H or C₁-C₆ alkyl; R¹² is C₁-C₆ alkyl;X is —(C═O)— or a direct bond; and a, b, c and d are each independentlyan integer from 1 to 24; wherein each methyl, alkyl and alkylene isindependently optionally substituted.
 2. The compound of claim 1, havingone of the following structures (IA) or (TB):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.3. The compound of claim 1, wherein G¹ is C₂-C₃ alkylene or C₄-C₆alkylene. 4-5. (canceled)
 6. The compound of claim 1, wherein G² isC₂-C₃ alkylene or C₃-C₄ alkylene.
 7. The compound of claim 1, wherein Xis —(C═O)—.
 8. The compound of claim 1, wherein X is a direct bond. 9.The compound of claim 1, wherein R⁷ is —O(C═O)R¹⁰ or —(C═O)OR¹⁰. 10.(canceled)
 11. The compound of claim 9, wherein R¹⁰ is linear C₆-C₁₀alkyl.
 12. The compound of claim 9, wherein R¹⁰ is methyl. 13.(canceled)
 14. The compound of claim 9, wherein R¹⁰ is branched C₁₀-C₁₅alkyl.
 15. The compound of claim 1, wherein R⁷ is —NR⁹(C═O) or—(C═O)NR⁹R¹⁰.
 16. The compound of claim 15, wherein R⁹ is H.
 17. Thecompound of claim 15, wherein R⁹ and R¹⁰ are each independently C₆-C₁₀alkyl. 18-21. (canceled)
 22. The compound of claim 1, wherein R^(1a),R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R^(4a) and R^(4b) are, at eachoccurrence, independently H or C₁-C₁₂ alkyl. 23-24. (canceled)
 25. Thecompound of claim 22, wherein at least one of R^(1b) and R^(4b) is C₁-C₈alkyl.
 26. (canceled)
 27. The compound of claim 1, wherein

or both, independently has one of the following structures:


28. The compound of claim 1, wherein a, b, c and d are eachindependently an integer from 2 to 12 29-32. (canceled)
 33. The compoundof claim 1, wherein R⁸ is —N(R¹¹)(C═O)R¹².
 34. The compound of claim 1,wherein R⁸ is —(C═O)NR¹¹R¹².
 35. The compound of claim 1, wherein R⁸ isNR¹¹R¹². 36-39. (canceled)
 40. The compound of claim 1, wherein R⁸ is—(C═O)OR¹².
 41. The compound of claim 40, wherein R⁸ is —O(C═O)R¹². 42.The compound of claim 1, wherein R⁸ has one of the following structures:


43. The compound of claim 1, having one of the following structures:


44. A lipid nanoparticle comprising the compound of claim 1 and atherapeutic agent. 45-58. (canceled)
 59. A pharmaceutical compositioncomprising the lipid nanoparticle of claim 44 and a pharmaceuticallyacceptable diluent or excipient.
 60. A method for treating or preventinga disease in a patient in need thereof, the method comprisingadministering the lipid nanoparticle of claim 44 to the patient.
 61. Amethod for vaccinating a patient in need thereof against a viralpathogen, the method comprising administering the lipid nanoparticle ofclaim 44 to the patient, wherein the therapeutic agent is a viralantigen or a nucleic acid capable of transcribing a viral antigen.